1. Field of the Invention
The present invention refers to a calibration aid for fluorescence measurement applications.
2. The Prior Art
An important field of application of fluorescence measurements is the field of non-invasive tissue perfusion quantification of a patient. A non invasive tissue perfusion imaging and quantification system is described in EP 1 210 906 A2. The patient is monitored with a digital camera while injecting indocyanine green (ICG, a fluorescent dye with an absorption maximum in the near infrared range) and irradiating the tissue of interest using a radiation source emitting radiation in the near infrared range. From the fluorescence signal of the tissue of interest (i.e. the light emitted by the fluorescent dye present in the blood flowing through the tissue) the perfusion state of the tissue can be determined using appropriate algorithms, such as disclosed in EP 1 210 906 A2. Equipment for intraoperative use of ICG measurements for determining tissue perfusion is further disclosed in DE 10059070 C1.
Advantageously, an external fluorescence standard is used when performing the measurements. It is placed next to the tissue of interest and, when irradiated, emits a constant and defined fluorescence signal which is recorded together with the fluorescence signal of the tissue of interest.
The use of a fluorescence standard allows to directly compare different measurements and to normalize measurement results based on the defined signal intensity of the fluorescence standard. It further allows compensating changes in the measurement conditions during a measurement (such as change in intensity of ambient light, change of exposure parameters of the camera or change of parameters of the radiation).
For reference purposes as described above, pure dry ICG dye is usually dissolved in water or methanol immediately before use. With this liquid sample an in vitro fluorescence measurement can be performed as described above. However, this practice is time consuming. Since ICG is not stable when exposed to air humidity and light, the reference standard must be prepared immediately before use, which is a major disadvantage when used in connection with urgent surgery. Moreover, the absorption and fluorescence properties of such pure dissolved standard samples are not equal to the properties of the protein bound dye after injection to a patient, e.g. the absorption maximum is shifted from 780 nm to 805 nm.
It is also not possible to use pure dry ICG dye for reference purposes as described above, since pure ICG powder also exhibits absorption and fluorescence properties different from dissolved ICG. Further, dry ICG may alter its properties due to ambient humidity.